biochemistry 2 chapter 10

what are enzymes

biological catalysts that increase reaction rates without being used up or affecting equilibrium

what are most enzymes in proper configuration

proteins, however some rna also catalyze reactions

what do some enzymes require

a cofactor
An ion such as Fe++, Mg++ or Zn++
An organic or organometallic coenzyme (e.g. heme, or NADP)
Holoenzyme is with the cofactor, apoenzyme is without the cofactor
Some cofactors are tightly bound (called prosthetic groups), others are loosely bound

how do enzymes affect equilibrium and thermodynamics

Enzymes do not affect the overall reaction equilibrium (Keq)
Enzymes do not alter the overall standard free energy change (change of G) of the reaction

mechanisms of rate increase by

Enzymes accelerate reactions exclusively by lowering the activation energy barrier for both the forward and reverse reactions

cofactor nature, binding, example, and activity role

inorganic or organic, loosely or tightly, mg2+ for DNA, stabilizes/activates

coenzyme nature, binding, example, and activity role

organic, usually loose, NAD/FAD/CoA, electron/group carriers

apoenzyme nature, binding, example, and activity role

protein only, none, Apo-DNA polymerase, inactive form

prosthetic group nature, binding, example, and activity role

organic or inorganic, tightly or covalently, heme in hemoglobin, permanent catalytic role

cofactor =

helper (metal ion or organic molecule)

coenzyme =

organic helper (vitamin derived)

apoenzyme =

inactive protein part alone

prosthetic group =

permanently bound helper

how do enzymes organize reactive groups

close proximity and proper orientation

uncatalyzed bimolecular reactions

two free reactants → single unrestricted transition state conversion is energetically unfavorable

uncatalyzed unimolecular reactions

flexible reactant → rigid transition state conversion is energetically unfavorable for flexible reactants

catalyzed reactions

the enzyme uses the binding energy of substrates to organize the reactant to a fairly rigid ES complex, the energy cost is paid during binding

What state do enzymes bind best to

transition states

enzyme active sites are complimentary to

the transition state of the reaction

enzymes bind transition states better than

substrates

stronger interactions with the transition state as compared with

the ground state lower the activation barrier

ion Fe2+ or Fe3+

enzymes Cytochrome oxidase, catalase, peroxidase

ion Mg2+

enzymes Hexokinase, glucose 6-phosphatase, pyruvate kinase

important coenzymes

biocytin, coenzyme A, flavin adenine dinucleotide, nicotinamide adenine dinucleotide, tetrahydrofolate

what is chymotrypsin

a serine protease that cleaves peptide bonds adjacent to aromatic amino acids (carboxyl side of Tyrosine, Phenylalanine, and Tryptophan)

what is a hydrophobic pocket

A deep binding pocket near the active site that selectively accommodates the bulky, nonpolar aromatic side chains (Tyr, Phe, Trp) of the substrate, properly positioning the adjacent peptide bond for cleavage.

what is the catalytic triad

Asp 102, His 57, Ser 195

His 57

Acts in general acid-base catalysis. It accepts a proton from Ser 195 to generate a strongly nucleophilic alkoxide ion (O-) and later donates a proton to the leaving group

Ser 195

Acts as a nucleophile. Its deprotonated alkoxide ion attacks the substrate peptide carbonyl carbon, forming a transient covalent acyl-enzyme intermediate

Asp 102

Hydrogen bonds with His 57 to orient and stabilize its positively charged form during catalysis

what is an oxyanion hole

A pocket formed by backbone amide hydrogen atoms (including Gly 193 and Ser 195).

what does an oxyanion hole do

It stabilizes the short-lived, negatively charged oxygen atom (O-) of the tetrahedral intermediates formed during nucleophilic attacks (acylation and deacylation phases).

step 1 of chymotrypsin mechanism

substrate binding to active site of chymotrypsin

step 2 of chymotrypsin mechanism

nucleophilic attack: histidine interacts with serine pulling H from Ser, generating nucleophile, the resulting alcoxide ion attacks peptide C=O of NH-C=O, forming tetrahedral intermediate (TH) with C-O formation

step 3 of chymotrypsin mechanism

substrate cleavage: collapse of TH intermediate reforms the C=O and breaks its bond with NH leaving first product and Acyl Enzyme intermediate

step 4 of chymotrypsin mechanism

water comes in gets deprotonated, forms nucleophile (OH-) and attacks the ester linkage of Acyl enzyme intermediate

step 5 of chymotrypsin mechanism

collapse of TH intermediate forms COOH on substrate breaking off from the enzyme giving the second product and serine

step 6 of chymotrypsin mechanism

break off from the enzyme

step 7 of chymotrypsin mechanism

product dissociates

Basic (histidine) and acidic (serine) functional groups on the enzyme act in

general acid base catalysis

Transient covalent bonds are formed between

the substrate and the enzyme

Chymotrypsin cleaves peptides on the carboxyl side of

tyrosine, phenylalanine and tryptophan because they contain aromatic rings, which fit into the hydrophobic pocket of the enzyme

kinetics definition

the study of the rate at which molecules react

what is the rate of enzymatic reaction affected by

enzyme, substrate, effectors, and temperature

competitive inhibitor mechanism

competes directly with the substrate for binding at the active site

competitive inhibitor kinetic impact

increases Km, while Vmax remains unchanged

uncompetitive inhibitor mechanism

binds only to the enzyme substrate complex, preventing the reaction from completing

uncompetitive inhibitor kinetic impact

decreases both Vmax and Km by the same factor

Noncompetitive inhibitor mechanism

binds to either the free enzyme or the enzyme substrate complex at a site distinct from the active site

Noncompetitive inhibitor kinetic impact

decrease Vmax, Km remains unchanged

total enzyme concentration is

constant

E+S→←

ES

ES→←

E+P

C is what in equation

E+S

U is what in equation

ES

Km

the substrate concentration at which the initial reaction velocity reaches half of the maximum velocity (1/2Vmax)

Why does Vmax changes affect Km

Km is anchored to the value of 1/2Vmax, so changes in Vmax directly influence the corresponding Km on the velocity curves

nonlinear michaelis menten plot should be used to

calculate parameters of Km and Vmax

a linearized double reciprocal plot is good for analysis of

two substrate data or inhibition

saturation kinetics

high S, velocity is NOT proportional to S

regulation can be

non covalent modification, covalent modification, irreversible, reversible

noncovalent modification

allosteric regulators are small chemicals that can be positive (improve) or negative (reduce)

what is ATCases primary role

to initiate the de novo pathway for pyrimidine nucleotide biosynthesis

what does ATCasae do

catalyzes the condensation of aspartate and carbamoyl phosphate to form N-carbamoylaspartate, in pyrimidine

allosteric behavior

It exhibits sigmoidal kinetics (saturation curve) rather than hyperbolic Michaelis-Menten kinetics, transitioning between two structural states

T state

low activity state

R state

high activity state

Allosteric regulators

ATP and CTP

ATP

positive modulator, allosteric activator.
Binds to ATCase and stabilizes the high-activity R state, shifting the curve to the left and lowering K

CTP

negative modulator, allosteric inhibitor.
Product of the pyrimidine pathway that acts as a feedback inhibitor. Binds to ATCase and stabilizes the low-activity T state, shifting the curve to the right and increasing

Methanol Poisoning

an application of competitive enzyme inhibition, where competitive inhibitors are used medically to block the active site of alcohol dehydrogenase to prevent the conversion of methanol into toxic metabolites

Aspirin

Highlighted as a key example of irreversible covalent modification (and suicide/irreversible inhibition), where the active site of the target enzyme is permanently modified to inhibit activity